Tbx18参与血管钙化的作用及机制研究

Vascular calcification is very common in a number of diseases including cardiovascular diseases, diabetes, and chronic kidney diseases. It is an independent risk factor for cardiovascular mortality and morbidity and heavily threatens human health. Tbx18 is an important transcription factor regulating cardiovascular development. Previous studies have shown that Tbx18 positive cells give rise to vascular smooth muscle cells during heart development. Our preliminary data also show that Tbx18 is highly expressed in calcified vascular smooth muscle cells, and knock-down of Tbx18 results in down-regulation of vascular calcification-related transcription factors including Runx2 and Sox9. Based on these findings, we hypothesize that Tbx18 may regulate vascular calcification through activation of Runx2 and Sox9. In vitro and in vivo model of vascular calcification will be used in this study. To investigate the role of Tbx18 in vascular calcification, we will use adenovirus encoding Tbx18 and short interfering RNA against Tbx18 to transfect vascular smooth muscle cells. We will also utilize gene knock-out technology in mice to study the function of Tbx18 in vascular calcification. Additionally, we plan to use RNA-Seq to identify potential downstream target genes of Tbx18. Interaction between Tbx18 transcription factor and downstream target genes will be studied by Chip-PCR, electrophoretic mobility shift assay and luciferase reporter analysis. Elucidation of mechanism underlying vascular calcification will provide a novel theoretical basis and treatment for vascular calcification.

血管钙化常见于心血管疾病，糖尿病和慢性肾病患者，是心血管疾病发生率和死亡率增加的独立危险因素，严重威胁人类健康。Tbx18是调节心血管系统发育的重要转录因子。既往研究发现Tbx18阳性细胞能分化成血管平滑肌细胞。我们的前期实验也表明Tbx18在钙化的血管平滑肌细胞中表达明显增高，且抑制Tbx18能下调血管钙化相关的重要转录因子Runx2和Sox9的表达。由此我们假设：Tbx18可能调节Runx2和Sox9参与血管钙化。本项目拟采用体内外相结合的血管钙化模型，运用腺病毒过表达，RNA干扰及基因敲除的方法，对Tbx18参与血管钙化的作用进行研究；应用RNA-Seq的方法，筛选Tbx18调控的下游基因，然后用染色质免疫共沉淀结合PCR，凝胶迁移，荧光素酶报告基因分析的方法研究转录因子Tbx18与下游基因的相互作用关系，阐明Tbx18调节血管钙化的分子机制，为血管钙化的防治提供新的理论依据和思路。

血管钙化是受细胞和基因调控的生物学过程，与骨生成过程类似。Tbx18 是调节心血管系统发育的重要转录因子。既往研究发现Tbx18可调节骨发育过程，但Tbx18是否调节血管钙化尚不清楚。在此基础上，本项目系统研究了Tbx18对血管钙化的调控作用及机制。我们首先研究了血管钙化过程中Tbx18的表达水平变化。结果显示：在人血管平滑肌细胞钙化过程中，Tbx18的表达水平升高；Tbx18在人体钙化血管呈现高表达。其次，我们研究了Tbx18对血管钙化的调节作用。细胞实验表明：过表达Tbx18可促进人血管平滑肌细胞钙化；敲低Tbx18的表达能明显减弱血管平滑肌细胞钙化。此外，过表达Tbx18能促进血管环钙化。进一步的动物实验证明过表达Tbx18能加速慢性肾病大鼠血管钙化，条件性敲除Tbx18可明显改善血管钙化。最后，我们阐明了Tbx18促进血管钙化的分子机制。结果显示：Tbx18可正向调控Sox9的表达，敲低Sox9可明显改善Tbx18诱导的血管平滑肌细胞钙化，提示Sox9介导了Tbx18诱导的血管钙化。综上所述，本研究证明了Tbx18可通过Sox9促进血管平滑肌细胞成骨样分化和血管钙化，为血管钙化的防治提供新的理论依据。